Quantitative testing for vitamin B12

ABSTRACT

The vitamin B 12  (cobalamin) level of human blood and mammalian tissue is routinely assayed utilizing radioisotope dilution assay (RIDA) techniques. The presence of vitamin &#34;B 12  analogues&#34; in most such samples, which analogues are erroneously assayed as true vitamin B 12  utilizing prior art RIDA techniques, has been determined. Such errors are due to the fact that binding proteins commonly used in prior art RIDA techniques usually include proteins which bind both true vitamin B 12  and the previously unrecognized vitamin B 12  analogues which are present in such samples. The errors previously caused by the measurement of vitamin B 12  analogues are avoided by using an assay composition for binding vitamin B 12  which is substantially free of substances which bind vitamin B 12  analogues in said samples. Methods of making and using such assay compositions are taught.

The invention described herein was made, in part, in the course of workunder a grant or award from the Department of Health, Education, andWelfare.

This is a continuation of application Ser. No. 893,524 filed Apr. 4,1978 now U.S. Pat. No. 4,188,189.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods and materials for assayingmammalian blood and tissue. More specifically it relates to methods andmaterials for determining the amounts of vitamin B₁₂ and vitamin B₁₂analogues in human plasma.

2. Prior Art

For many years it has been recognized that the assay of the vitamin B₁₂level in humans is a valuable technique for diagnosing and subsequentlytreating certain diseases, such as for example, pernicious anaemia, postgastrectomy states nutritional deficiencies, intestinal disorders, andothers. Initially, vitamin B₁₂ was assayed microbiologically usingeither Euglena gracilis or Lactobacillus leichmannii. More recently,radioisotope dilution (RID) assays for B₁₂ have been utilized. Suchradioisotope dilution assay techniques are well documented in theliterature, see for example Lau, et.al. (1965) "Measurement of Serum B₁₂Levels Using Radioisotope Dilution and Coated Charcoal," BLOOD, 26, 202,as modified by Raven et.al. (1968) "The Effect of Cyanide Serum andOther Factors on the Assay of Vitamin B₁₂ by Radio-Isotope Method Using⁵⁷ Co-B₁₂, Intrinsic Factor and Coated Charcoal," GUYS HOSPITAL REPORTS,117, 89; and (1969) "Improved Method for Measuring Vitamin B₁₂ in SerumUsing Intrinsic Factor, ⁵⁷ Co-B₁₂ and Coated Charcoal," JOURNAL OFCLINICAL PATHOLOGY 22, 205.

Such prior art radioisotope dilution assay of vitamin B₁₂ generallyincludes the steps of freeing the endogenous B₁₂ from its naturalbinding protein by boiling at a selected pH and then adding a measuredamount of the radioisotope ⁵⁷ Co-B₁₂ and a limited amount of bindingprotein. All of the binding protein will be bound by some form of B₁₂since the amount of radioisotope B₁₂ added is, by itself, sufficient tobind the small amount of protein. As both the natural B₁₂ and theradioactive B₁₂ compete to bind with the protein, the degree to whichthe radioactive count of the protein bound B₁₂ was inhibited was thoughtto be indicative of the amount of natural B₁₂ present in the sampleundergoing testing.

More specifically, in the technique of Lau et.al. as modified by Ravenet.al., serum B₁₂ is separated from binding protein in the plasma sampleby boiling with 0.25 N HCl. Radioisotope B₁₂ is added to the reactionmixture and the B₁₂ mixture is reacted with protein, normally in theform of a commercially available binder. Then the free or unbound B₁₂ isseparated from the protein bound B₁₂ by protein-coated charcoal and theradioactivity of the supernatant liquid containing the mixture of boundradioactive B₁₂ and bound non-radioactive B₁₂ counted for radioactivity.The serum B₁₂ concentration is then calculated from the count, often bycomparison with a standard chart. Almost as soon as this technique beganto be utilized it was recognized that the vitamin B₁₂ measurements itprovided were usually inconsistent with the results obtained by othermeasuring techniques for B₁₂, such as the microbiological assays. Mostoften, the vitamin B₁₂ assay obtained by radioisotope dilutiontechniques have been found to be high. Many theories have been advancedto explain the cause of the high vitamin B₁₂ readings. However, it isbelieved that nowhere in the prior art is it recognized that there aresubstances in mammalian blood and tissue which react with certainnon-specific protein binders in the radioisotope dilution assaytechnique to provide an analysis of vitamin B₁₂ which is apparentlyhigher than the amount of B₁₂ actually in the sample. Additionally, itis believed that nowhere in the prior art is it recognized that mostcommon and commercial RID assay protein binders are not specific tovitamin B₁₂, but that they are also capable of binding with theheretofore unknown B₁₂ analogues and thus provide erroneous B₁₂ assays.

BRIEF DESCRIPTION OF THE INVENTION

As has already been indicated, in the standard radioisotope bindingassay for vitamin B₁₂, a known amount of radioactive vitamin B₁₂ ismixed with a prepared to-be-tested sample. Then, a known, but extremelylimited, amount of protein which is capable of binding with both thenatural and radioactive vitamin B₁₂ is added to the mixture. Then,utilizing well known techniques, the radioactivity of the bound sampleis compared, for example, with a standard curve to determine the amountof natural vitamin B₁₂ present in the tested sample. Such standardcurves are initially established for use in RID assay, for example, bymeasuring the amount of bound radioactive B₁₂ in the presence of thesame type and amount of protein binder, but with several differentamounts of known non-radioactive B₁₂.

It has now been discovered, for what is believed to be the first time,that mammalian blood and tissue contain materials other than vitamin B₁₂which couple with certain binding proteins which are commonly used inRID assays. For purposes of this specification and claims thenon-vitamin B₁₂ materials which are capable of binding with suchproteins will be herein referred to as "vitamin B₁₂ analogues," "B₁₂analogues" or simply as "analogues." They are referred to as analogues,not due to their chemical structure, which is not known with certainty,nor in the commonly accepted chemical sense of the word "analogue."Rather they are referred to as analogues due to their reactivity withthe binding proteins commonly used in RID assays. As will be shown inmore detail, hereinafter, there are other similarities which have beendiscovered between vitamin B₁₂ and the newly discovered analogues whichare preent in mammalian blood and tissue.

After the presence of B₁₂ analogues was discovered it was thendetermined that protein binders commonly present in RID assays were: (1)Non-specific in binding to only vitamin B₁₂ ; and (2) reactive inbinding with both vitamin B₁₂ and B₁₂ analogues; and (3) capable ofreacting with both B₁₂ and B₁₂ analogues independent of pH. These aremost commonly R proteins. Additionally, it has been determined thatother protein binders, are: (1) Very specific in their reactivitysubstantially only with vitamin B₁₂ ; (2) substantially non-reactivewith the B₁₂ analogues; and (3) non-reactive with either vitamin B₁₂ orB₁₂ analogues in highly acid environments. These are most commonlyproteins in the form of pure human intrinsic factor (IF). hog IF, rabbitIF, other IFs and vitamin B₁₂ specific binders.

In the past the problem has been that RID binders include substantialamounts of protein which is not specific to vitamin B₁₂. Therefore, theradioisotope dilution assay utilizing that binder on samples whichcontain B₁₂ analogues will produce a measurement which indicates agreater amount of B₁₂ present in the plasma than exists in fact. As willbe shown in more detail hereinafter, commercially available proteinbinders, which have heretofore been labeled as containing intrinsicfactor, in fact include only about 10% to about 30% intrinsic factorprotein, while the balance of the protein in the binder is of anon-specific type, such as R protein. Thus, the protein materials in thecommercial protein binders are capable of indiscriminate reaction withthe heretofore unrecognized vitamin B₁₂ analogue materials in mammalianblood and tissue. These extraneous reactions give RID analyses havingthe appearance of apparently higher vitamin B₁₂ content than the samplesin fact contain. This is due to the fact that when the binder includesprotein which is non-specific to vitamin B₁₂ and which is capable ofreacting with both vitamin B₁₂ and B₁₂ analogues, then the use of thisprotein in the radiobinding assay measures both the vitamin B₁₂ and thevitamin B₁₂ analogues which are present in the sample. However, inaccordance with the present invention, when the proteins which areutilized are substantially specific to vitamin B₁₂, such assubstantially pure intrinsic factor, then in the RID assay one binds andmeasures substantially only the vitamin B₁₂ in the sample, without themeasurement of extraneous B₁₂ analogues. This provides a more accuratevitamin B₁₂ RID assay.

Based on these discoveries it is proposed that in the practice of RIDassay only protein which is specific in its reaction to vitamin B₁₂ beutilized. Alternatively, it is proposed that mixtures of vitamin B₁₂specific and non-specific binding proteins be treated, for example, withan excess of material such as B₁₂ analogues, which will bind orinactivate only the non-specific binding proteins, prior to thematerials use in RID assays, so that the non-specific protein will besubstantially unavailable for reaction with any vitamin B₁₂ or analoguesin a sample when the RID assay is conducted. In yet another modificationof the present invention, crude binder, including non-specific bindingproteins, is subjected to proteolytic enzyme treatment prior toutilization as a vitamin B₁₂ binder in RID assays. Such proteolyticenzyme treatment destroys the binding ability of the non-specificproteins without destroying the binding ability of the proteins whichare specific to vitamin B₁₂.

Utilizing the techniques of the present invention, the B₁₂ analogues canbe assayed by analyzing the amount of vitamin B₁₂ present utilizing, forexample, a vitamin B₁₂ specific binder, then assaying the sampleutilizing a non-specific binder and determining the difference betweenthe two assays as a measure of the amount of vitamin B₁₂ analoguespresent.

These and other techniques are readily determined, once, as taught forthe first time by the present invention, the presence of B₁₂ analoguesin mammalian blood and tissue is recognized.

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the following examples and tables certain chemical components wereutilized. For ease of communication they have been given shortened namesin the text. The concordance between the "component" names and theiractual compositions is as follows:

    ______________________________________                                        Components                                                                              Actual Composition                                                  ______________________________________                                        A. Buffer 1.0M Tris (hydroxymethyl) aminomethane-                                       HCl pH 10.0                                                         B. Albumin                                                                              Bovine serum albumin, 2 mg per ml in H.sub.2 O                      C. Salt   0.15M NaCl                                                          D. Boiled                                                                       buffer  (1 part) 0.5M sodium acetate-HCl pH 4.5                                       (1 part) 0.01M KPO.sub.4 pH 7.5, 0.15M NaCl                                   (2 parts) 50 μg per ml KCN in 0.15M NaCl                                   The complete solution is heated for 45 min.                                   at 100° C.                                                   E. Standard                                                                     (100 pg/ml                                                                            Solution D containing 100 pg per ml vitamin                           B.sub.12)                                                                             B.sub.12. The solution is heated for 45 min. at                               100° C. after the vitamin B.sub.12 is added. The                       concentration of vitamin B.sub.12 in the stock                                solution used to make component E is deter-                                   mined by its light absorbance at 278, 361                                     and 550 nm.                                                         F. Standard                                                                     (1000 pg/ml                                                                           Same as component E except that the vitamin                           B.sub.12)                                                                             B.sub.12 concentration is 1000 pg/ml.                               G. (.sup.57 Co)                                                                 B.sub.12                                                                              1000 pg per ml of (.sup.57 Co) B.sub.12, (150-300                             μCi/μg), in H.sub.2 O.                                        H. Binder Present in 0.01M Tris-HCl pH 8.2, containing                                  0.15M NaCl and 50 μg per ml bovine serum                                   albumin. Binders are diluted in this solu-                                    tion to reach a concentration of 700 pg per                                   ml of vitamin B.sub.12 binding ability.                                       Individual binders are as follows:                                            (1) Human intrinsic factor (Human IF) -                                       Human gastric juice containing more                                           than 95% intrinsic factor based on                                            assays employing inhibition of vitamin                                        B.sub.12 binding with anti-intrinsic factor                                   antibodies (>95% inhibition) and co-                                          binamide (<5% inhibition).                                                    (2) Human R protein (Human R) - Human saliva                                  containing more than 95% R protein based                                      on assays employing inhibition of vita-                                       min B.sub.12 binding with cobinamide (>95%                                    inhibition) and anti-intrinsic factor                                         antibodies (<5% inhibition)                                                   (3) Hog intrinsic factor (Hog IF) - This                                      protein was purified from "Hog in-                                            trinsic factor concentrate" by affinity                                       chromatography on vitamin B.sub.12 -Sepharose                                 employing gradient elution with guani-                                        dine-HCl followed by gel filtration.                                          The final preparation contained more                                          than 95% intrinsic factor based on                                            assays employing inhibition of vitamin                                        B.sub.12 binding with anti-intrinsic factor                                   antibodies (>95% inhibition) and co-                                          binamide (<5% inhibition).                                                    (4) Hog R protein (Hog R) (Also designated                                    in the scientific literature as Hog non-                                      intrinsic factor) - This protein was                                          purified from "Hog intrinsic factor                                           concentrate" as described above in (3).                                       The final preparation contained more                                          than 95% R protein based on assays em-                                        ploying inhibition of vitamin B.sub.12 bind-                                  ing with anti-intrinsic factor antibod-                                       ies (<5% inhibition) and cobinamide                                           (>95% inhibition).                                                            (5) Rabbit intrinsic factor (Rabbit IF) -                                     An extract of rabbit gastric mucosa                                           containing more than 95% intrinsic fac-                                       tor based on assays employing inhibition                                      of vitamin B.sub.12 binding with anti-intrin-                                 sic factor antibodies (>95% inhibition)                                       and cobinamide (<5% inhibition).                                              (6) Hog intrinsic factor concentrate (Hog                                     IFC) - A crude extract of hog pyloric                                         mucosa. It contained 25% Hog IF and                                           75% Hog R based on assays employing                                           inhibition of vitamin B.sub.12 binding with                                   anti-intrinsic factor antibodies (25%                                         inhibition) and cobinamide (75% inhib-                                        tion).                                                                        (7) Hog IFC + Cobinamide - Hog IFC contain-                                   ing the vitamin B.sub.12 analogue cobinamide                                  ([CN, OH]Cbi) in a molar amount equal                                         to 100 times the total vitamin B.sub.12                                       binding ability, i.e. a 100 fold excess                                       of cobinamide.                                                      The same as8) Hog IFC + CN-Cbl[bde-OH]                                                  item (7) above except that the analogue                                       added is CN-Cbl[bde-OH] and is present                                        in a 1000 fold molar excess.                                                  (9) Hog IFC +                                                                 [3,5,6-Me.sub.3 BZA](CN, OH)Cba -                                             The same as item (7) above except that                                        the analogue added is [3,5,6-Me.sub.3 BZA]                                    (CN, OH) Cba.                                                                 (10) Digested Hog IFC - Hog IFC incubated                                     with bovine pancreatic trypsin (2 mg                                          per ml) and bovine pancreatic chymo-                                          trypsin (2 mg per ml) for 60 min. at                                          37° C.                                                       I. Charcoal                                                                             A solution containing 25 mg per ml neutral                                    charcoal (Norit) and 5 mg per ml bovine                                       serum albumin in H.sub.2 O.                                         J. unknown                                                                      sample  Samples containing unbound vitamin B.sub.12 are                               diluted in solution D (see above). Samples                                    containing bound vitamin B.sub.12, such as serum,                             are prepared as follows:                                                      (1 part) sample                                                               (1 part) 0.5M sodium acetate-HCl pH 4.5                                       (2 parts) 50 μg per ml KCN in 0.15M NaCl                                   The complete mixture is heated at 100° C.                              for 45 min. followed by centrifugation                                        at 5000 × g                                                             at 4° C. for 20 min. The super-                                        natant is removed and used for assay.                               ______________________________________                                    

Each of the RID assays referred to herein utilized the componentsreferred to above. The method and order of utilizing the components isthat set forth in Table 1. That is, components A, B, C, etc. or thebuffer, albumin, and salt, respectively, etc. were added in the order,from left to right, shown in Table 1.

                                      TABLE I                                     __________________________________________________________________________    Flow Sheet for Radiobinding Assay for Vitamin B.sub.12                                          D   E      F       J                                              A   B    C  Boiled                                                                            Standard                                                                             Standard                                                                              Unknown                                                                             G     H   I                              Buffer                                                                            Albumin                                                                            Salt                                                                             Buffer                                                                            100pg/ml B.sub.12                                                                    1000pg/ml B.sub.12                                                                    Sample                                                                              [.sup.57 Co] B.sub.12                                                               Binder                                                                            Charcoal                 Tube #                                                                              (ul)                                                                              (ul) (ul)                                                                             (ul)                                                                              (ul)   (ul)    (ul)  (ul)  (ul)                                                                              (ul)                     __________________________________________________________________________    1,2   225 50   875                                                                              800 --     --      --    50    --  --                       3,4   225 50   375                                                                              800 --     --      --    50    --  500                      5,6   225 50   325                                                                              800 --     --      --    50    50  500                       7    225 50   325                                                                              720  80    --      --    50    50  500                       8    225 50   325                                                                              640 160    --      --    50    50  500                       9    225 50   325                                                                              560 240    --      --    50    50  500                      10    225 50   325                                                                              300 500    --      --    50    50  500                      11    225 50   325                                                                              720 --      80     --    50    50  500                      12    225 50   325                                                                              640 --     160     --    50    50  500                      13    225 50   325                                                                              450 --     350     --    50    50  500                      14    225 50   325                                                                              --  --     800     --    50    50  500                      Unknown                                                                       Sample                                                                              225 50   325                                                                              --  --     --      800   50    50  500                      __________________________________________________________________________

After the addition of ⁵⁷ Co-B₁₂ the components are mixed thoroughly tomix both the naturally occurring B₁₂ and the radioisotope B₁₂ to makethem compete and equally available to react with the binder. After theaddition of the binder, H, the components were again mixed thoroughly,and then incubated for 45 minutes at about 37° C. Charcoal was thenadded to the incubated mixture and the components again mixed thoroughlyand incubated for another 5 minutes at room temperature. This wasfollowed by centrifuging at 2000×g at 4° C. for 30 minutes. Then 1000 ulof the resulting supernatant liquid is pipetetted from the sample and adetermination of the amount of ⁵⁷ Co-B₁₂ present is made. The amount of⁵⁷ Co-B₁₂ is indicative of the amount of natural B₁₂ in the testedsample, with lesser amounts of ⁵⁷ Co-B₁₂ being indicative of greateramounts of natural vitamin B₁₂ in the sample.

Calculations of vitamin B₁₂, utilizing the data obtained in theforegoing manner, is made as follows:

Calculation of data from radiobinding assay for plasma vitamin B₁₂ assayas outlined in Table I

(1) The values in tubes 3 and 4, the "blank" tubes without binder areaveraged and subtracted from all other tubes starting with tube 5.

(2) The background radiation is subtracted from tubes 1 and 2 and thesevalues are averaged.

(3) Tubes 5 and 6 are averaged. This value should be at least 15% belowthe average value for tubes 1 and 2 to insure that all of the binder issaturated in the presence of (⁵⁷ Co) B₁₂ alone.

(4) Values for each tube beginning with tube 7 are divided by theaverage of tubes 5 and 6 to give values for "% trace binding."

(5) Percent trace binding for tubes 7-14 are used to obtain a standardcurve. We plot % trace binding on the ordinate of logit-log paper versuspg vitamin B₁₂ on the log scale.

(6) The amount of vitamin B₁₂ in unknown samples is determined byinterpolation from the standard curve or data of % trace binding versuspg vitamin B₁₂.

(7) The standard curves for all of the various binders used arevirtually indistinguishable and vary little from day to day.Nevertheless, a complete standard curve is always obtained for everybinder with each set of assays. Representative data obtained with theassays are present in Table II.

                  TABLE II                                                        ______________________________________                                        Standard Curves obtained with the radiobinding assay for                      vitamin B.sub.12 using various binders                                                  % trace binding                                                     Vitamin B.sub.12                                                                        observed with various binders.sup.(b)                               added.sup.(a)                                                                           Human     Human   Hog    Hog  Hog                                   (pg)      IF        R       IF     R    IFC                                   ______________________________________                                        0         (100)     (100)   (100)  (100)                                                                              (100)                                 8         90        93      94     89   89                                    16        85        85      83     83   80                                    24        79        78      74     76   76                                    50        64        60      58     56   59                                    80        45        44      44     43   43                                    160       30        28      28     27   28                                    350       15        14      15     14   14                                    800        6         6       7      6    7                                    ______________________________________                                         .sup.(a) The vitamin B.sub.12 was boiled for 45 min. at 100° C. in     the same solution used to extract endogenous vitamin B.sub.12 for human       plasma.                                                                       .sup.(b) Assays were performed on different days.                        

Evidence as to the Origin and Existence of Vitamin B₁₂ Analogues inMammalian Blood and Tissue

Once the problem of the prior art is recognized, that is, that there arevitamin B₁₂ analogues present in mammalian blood and tissue, it becomesa relatively simple matter to prove the existence and chemistry of suchanalogues. It is also appropriate to prove that the various steps of theRID assay do not cause the B₁₂ analogues to be formed, for example, fromvitamin B₁₂.

In one instance this has been most convincingly shown by obtaining purecrystalline vitamin B₁₂, subjecting various known concentrations of itto the same conditions used to extract endogenous vitamin B₁₂ from bloodand tissue samples (boiling for 45 minutes in the same extractionsolution) and then analyzing them by RID assay using several bindingproteins, for example, in the form of human IF, hog IF, human R, hog Rand hog IFC on different portions of the same extracted vitamin B₁₂samples.

Referring to Table II, it will be seen that when various known amountsof pure vitamin B₁₂, ranging from about 8 pg to about 800 pg were testedwith various protein binders, that in each instance, the percent ofradioactive trace binding, or more accurately, the inhibition of ⁵⁷Co-B₁₂ binding, observed was substantially the same for each binder. Itis thus seen, that regardless of which protein binder is utilized, thepercent binding, i.e. inhibition of the (⁵⁷ Co)-B₁₂ is substantially thesame. This is indicative of the fact that during preparation for RIDassay the pure vitamin B₁₂ was not converted to analogues of the typewhich have now been observed in mammalian blood and tissue. It is alsoindicative of the fact that in the absence of interferring maskingcomponents in the samples, such as B₁₂ analogues, any of the bindingproteins can be utilized to provide substantially equally accurate RIDassays of vitamin B₁₂. Furthermore, the data in Table II should besuitable as a standard in the determination of vitamin B₁₂ by the sameRID assay.

By comparison, when endogenous vitamin B₁₂ was extracted from serum from74 normal blood donors (37 women, 37 men, ages 17-61) and testedutilizing the same binding proteins with the exception of hog IFC whichwas not used, the results were quite different. In every case in whichserum from normal donors was tested greater inhibition of ⁵⁷ CO-B₁₂, andtherefore greater apparent vitamin B₁₂, was observed with assaysemploying, as the binder, human R or hog R than was observed with assaysemploying human IF or hog IF. The data on the 74 normal donors isincluded in Table III.

                                      TABLE III                                   __________________________________________________________________________    Distribution of serum vitamin B.sub.12 values as measured with various        binders for 74 normal subjects and 21 patients                                with clinical evidence of vitamin B.sub.12 deficiency                         All assays were performed at pH 9.0                                           Serum  Human R Hog R                       Human R minus                                                                           Hog R minus              Vitamin B.sub.12                                                                     Nor-    Nor-    Human IF  Hog IF    Human IF  Hog IF                   (pg/ml)                                                                              mals                                                                             Patients                                                                           mals                                                                             Patients                                                                           Normals                                                                            Patients                                                                           Normals                                                                            Patients                                                                           Normals                                                                            Patients                                                                           Normals                                                                            Patients            __________________________________________________________________________     0-24                       8         11   1         2                        25-49                       6         5              1                        50-74                       5         3         1    2    1                   75-99     1       1         2         2    2                                  100-124   2       1                        1    3    9    3                   125-149   2       5    4         1         7    3    5    3                   150-174   3       1    5         5         6    5    9    5                   175-199   4       3    2         3         6    3    8    3                   200-224                                                                              1  1       2    5         4         8    2    11   2                   225-249   3    1  1    6         4         2    3    6    3                   250-274           3    10        9         10        4                        275-299   1    2  1    4         3         5         1                        300-324                                                                              1  2    4  2    12        4         6         3                        325-349                                                                              3  1    2  1    5         6         4    1    5    1                   350-374                                                                              5  1    1                 10        4         1                        375-399                                                                              3       2       3         4         4         2                        400-424                                                                              5       3       4         5         5         1                        425-449                                                                              3       4       3         2         2                                  450-474                                                                              4       6       2         3                   1                        475-499                                                                              5       5                 3                   1                        500-524                                                                              7       4       1         3                                            525-549                                                                              3       4       1                                                      550-574                                                                              5       3       2         1                   1                        575-599                                                                              1       2       1                   1                                  600-624                                                                              2       4       1         3                   1                        625-649                                                                              5       4                                                              650-674                                                                              3       3       2                                                      675-699                                                                              2       3                                                              700-724                                                                              3       6                 1                                            725-749                                                                              2                                                                      750-774                                                                              3       1                 1                                            775-799        1       1                                                      800-824        2                                                              825-849                                                                       850-874                                                                              1       1                                                              875-899                                                                              1       2                 1                                            900-924                                                                              1       1                                                              925-949                                                                              2       1                                                              950-974                                                                              1                                                                      975-999                                                                              1                                                                      1000-1024                                                                     1025-1049                                                                     1050-1074                                                                            1                                                                      1075-1099                                                                     1100-1124                                                                     1125-1149      1                                                              1150-1174                                                                     1175-1199                                                                     1200-1224                                                                     1225-1249                                                                            1                                                                      range of                                                                             220-                                                                             85-355                                                                             245-                                                                             84-342                                                                             130-785                                                                            0-78 135-880                                                                            0-86 12-575                                                                             56-347                                                                             0-605                                                                              72-342              serum B.sub.12                                                                       1230    1135                                                           mean of                                                                              548     542     298       336       237       179                      serum B.sub.12.sup. (a)                                                       mean ± 2                                                                          203-    197-    136-656   157-717                                      Std. Dev.                                                                            1482    1493                                                           __________________________________________________________________________     .sup.(a) Based on values obtained with log vitamin B.sub.12. Log values       were used since these gave a normal curve while the curve using               untransformed values were skewed to the right.                           

Other data concerning patients with diagnosed vitamin B₁₂ deficienciesare present, and comparisons between the normal donors and patients havealso been made on Table III, and will be discussed in more detailhereinafter. Referring to Table III it is seen that the mean endogenousvitamin B₁₂ RID assay levels, in terms of pg of vitamin B₁₂ per ml ofserum, are 548 and 542 for human R and hog R, respectively, but only 298and 361 for human IF and hog IF, respectively. This demonstrates thatsomething is present in extracts of normal human serum which inhibitsthe vitamin ⁵⁷ Co-B₁₂ binding ability of human R and hog R to a greaterextent than it inhibits the binding ability of human IF and hog IF.Under current RID assay techniques the greater inhibition which is foundusing human R and hog R is analyzed to indicate a higher vitamin B₁₂content. It is those substances, which have now been found to be presentin human blood serum and which preferentially inhibit ⁵⁷ Co-B₁₂ bindingof human R and hog R, which have been herein denominated as "vitamin B₁₂analogues."

Chemical Nature and Properties of Vitamin B₁₂ Analogues

The vitamin B₁₂ analogues, which are herein for the first timeidentified as being present in mammalian blood and tissue, have beenisolated by paper chromatography and compared with pure vitamin B₁₂.Vitamin B₁₂ and the so-called "vitamin B₁₂ analogues" were found to havethe following properties in common: (1) Both were adsorbed to charcoaland remained adsorbed when the charcoal was washed with 5% phenol; (2)Both were eluted from charcoal when the charcoal was washed with 67%acetone; (3) Both were extracted from aqueous solution into phenol andremained in the phenol phase even when the phenol was washed repeatedlywith water; (4) Both passed into the aqueous phase when the phenol layerwas dissolved in an excess of diethyl ether; (5) Both eluted withsimilar apparent molecular weights (approximately 1356) during gelfiltration on columns of Bio-Rad P-4 polyacrylamide; (6) Both wereadsorbed to a column of Sepharose-2B agarose that contained covalentlybound hog R protein and both remained bound when the column was washedwith 0.1 M glycine-NaOH pH 10.0, 1.0 M NaCl, and both were eluted fromthe Sepharose with either 85% phenol or 60% pyridine. Because of thesesimilarities the newly discovered material is seen to be similar tovitamin B₁₂ and is thus referred to as vitamin B₁₂ analogue.

The chemical nature and structure of the newly discovered vitamin B₁₂analogues which are now found to be present in mammalian blood andtissue is not known. An effort was made to compare them with chemicallytrue forms of vitamin B₁₂, sometimes referred to in the literature asanalogues of vitamin B₁₂, namely CN-B₁₂, OH-B₁₂, adenosyl-B₁₂ and CH₃-B₁₂, already known to be present in serum and tissues. This was done byadding 500 pg of each of these four known forms of vitamin B₁₂ to fourdifferent portions of the same human serum, in the dark. Prior to theadditions the serum contained 250 pg and 450 pg of vitamin B₁₂ asassayed by RID using human IF and human R, respectively, thus exhibitinga difference of 200 pg. After addition of the materials to the serum,each was allowed to incubate in the dark for 15 minutes to allow bindingof the added known forms of vitamin B₁₂ to the binding proteins normallypresent in the serum. Then the serum, with the added forms of vitaminB₁₂ was extracted utilizing standard conditions and the apparent amountof vitamin B₁₂ assayed by RID utilizing both human R protein and humanIF protein. Both the human R and human IF assays showed an increase inthe apparent amount of vitamin B₁₂ of about 500 pg. However, theoriginal difference observed between the values obtained with the humanR protein and the human IF protein, i.e. 200 pg, did not change. If anyof the added known forms of vitamin B₁₂ in the human serum had beenconverted to the newly discovered analogues, then the assays would haveshown an increase in the difference. This provides evidence that thenewly discovered vitamin B₁₂ analogues were not formed from any of theknown endogenous forms of native vitamin B₁₂ during the extractionprocedure.

Isolation of Vitamin B₁₂ Analogue

The materials which are herein designated as "vitamin B₁₂ analogues" andwhich have been found to preferentially inhibit R proteins in thevitamin B₁₂ assays were substantially separated from endogenous vitaminB₁₂ by the following purification scheme. A trace amount of 150 pg ⁵⁷Co-B ₁₂, was added to 1800 ml of freshly collected normal human plasma.The added ⁵⁷ Co-B₁₂ was sufficiently small that it did not interferewith subsequent RID assays. After incubating at room temperature for 30minutes the vitamin B₁₂ was extracted and assayed under standardconditions. When human IF binder was utilized in the RID assay theextract was found to contain 1050 ng of vitamin B₁₂, but when human Rprotein was utilized as the binder it appeared to contain 2030 ng ofvitamin B₁₂, almost twice as much vitamin B₁₂. The extract was thenpassed through a column of Sepharose containing covalently bound hog Rprotein. The column retained greater than 99% of the ⁵⁷ Co-B₁₂ as wellas the endogenous vitamin B₁₂ as assayed by RID with human IF or human Rprotein. After the column was washed with a variety of buffers and waterthe material was eluted with 60% pyridine, taken to dryness undervacuum, dissolved in water, and adsorbed onto charcoal. The charcoal waswashed with 5% phenol followed by water and the mixture of vitamin B₁₂,⁵⁷ Co-B₁₂ and analogue B₁₂ was eluted from the charcoal with 67%acetone. The material was again taken to dryness under vacuum, dissolvedin water, and then separated utilizing 19 inch long Whatman 3 MM paperfor paper chromatography and a solvent system consisting of 800 mlsec-butanol, 8 ml glacial acetic acid, 6 mmol HCN and 400 ml water. Thechromatography was performed in the descending manner for 30 hours atroom temperature in an environment that inhibited evaporation of thesolvent. The paper chromatogram was allowed to dry in a fume oven anddivided into 38 one-half inch fractions and numbered, with fraction 1starting at the point of application and number 38 being at the lowestpoint on the chromatogram. Each one-half inch fraction was thenincubated with 5 ml of water at 4° C. for twelve hours to elute thevitamin B₁₂, ⁵⁷ Co-B₁₂ and B₁₂ analogues. The water was then removed andtaken to dryness under vacuum. Each dried fraction was then dissolved in2.5 ml of water and assayed for ⁵⁷ Co-B₁₂ and for vitamin B.sub. 12using a variety of binding proteins. The final recovery of ⁵⁷ Co-B₁₂ was64%. The apparent recoveries of vitamin B₁₂ were 75% when using human IFin the assay and 66% when using human R in the assay. The results of theassays employing the 38 fractions obtained by paper chromatography arepresented in Tables IVA, IVB, and IVC. Similar data concerning paperchromatography of ⁵⁷ Co-B₁₂ and pure vitamin B₁₂, for reference as acontrol, are presented in Table IVD. The data in these severalchromatography tables summarized for convenience in Table IVE revealsthat the behavior of ⁵⁷ Co-B₁₂ that was extracted from human plasma didnot change its chromatographic behavior, and thus was not altered duringthe standard extraction procedure or any of the purification steps. In asimilar manner it is postulated that true vitamin B₁₂ is not altered inany of the purification or process steps of the assay.

                                      TABLE IVA                                   __________________________________________________________________________     [.sup.57 Co]  B.sub.12 and plasma B.sub.12 assayed after elution from        paper chromatography.                                                         All assays were performed at pH 9.0 except where indicated.                                B.sub.12 assayed with various binders                                               pH 1.8      pH 1.8                                         [.sup.57 Co] Human R12                                                                           Human R                                                                             Hog R Hog R Hog IFC                                  Fraction #                                                                          (cpm)                                                                             (%)                                                                              (ng)                                                                             (%)                                                                              (ng)                                                                             (%)                                                                              (ng)                                                                             (%)                                                                              (ng)                                                                             (%)                                                                              (ng)                                                                             (%)                                   __________________________________________________________________________    1            2     1     1     1     1                                        3                                                                             4            1     1     1     2     1                                        5            2           2     3     2                                        6            3     3     2     4     2                                        7            4     4     4     5     3                                        8            4     3     4     5     3                                        9            7  1  6     6  1  8  1  5  1                                     10    300 1  24 2  29 2  26 2  29 2  16 2                                     11    500 2  42 3  54 4  36 3  49 3  26 3                                     12    300 1  25 2  34 2  26 2  38 2  19 2                                     13    200 1  31 2  44 3  30 3  55 4  22 2                                     14    4600                                                                              20 209                                                                              16 235                                                                              16 176                                                                              16 240                                                                              16 180                                                                              17                                    15    14000                                                                             61 562                                                                              42 615                                                                              43 461                                                                              41 576                                                                              37 474                                                                              46                                    16    2100                                                                              9  103                                                                              8  122                                                                              9  84 7  122                                                                              8  91 9                                     17    300 1  85 6  118                                                                              8  85 8  128                                                                              8  53 5                                     18    300 1  68 5  68 5  68 6  100                                                                              6  45 4                                     19    200 1  23 2  17 1  17 2  27 2  15 1                                     20           45 3  39 3  39 3  60 4  24 2                                     21           42 3  25 2  25 2  52 3  21 2                                     22           8  1  5     7  1  8  1  5  1                                     23           8  1  4     5     8  1  4                                        24           7  1  4     5     7     3                                        25           9  1  1     6  1  3     4                                        26           16 1  1     10 1  4     5  1                                     27           5     1     3     2     2                                        28           2           1     1     1                                        29           4           2     1     2                                        30           1           1           1                                        31           1                                                                32                                                                            33                                                                            34                                                                            35                                                                            36                                                                            37                                                                            38                                                                            __________________________________________________________________________

                  TABLE IVB                                                       ______________________________________                                        Assay of [.sup.57 Co] B.sub.12 and plasma B.sub.12                            after elution from paper chromatography.                                      All assays were performed at pH 9.0.                                                       B.sub.12 assayed with various binders                            [.sup.57 Co] B.sub.12                                                                        Human IF  Hog IF    Rabbit IF                                  Fraction #                                                                            (cpm)   (%)    (ng) (%)  (ng) (%)  (ng) (%)                           ______________________________________                                        1                      1                   1                                  3                                                                             4                                                                             5                                1         1                                  6                                1                                            7                                1         1                                  8                                1         1                                  9                      1         1         2                                  10      300     1      4    1    5    1    8    1                             11      500     2      7    1    10   1    16   2                             12      300     1      5    1    7    1    10   1                             13      200     1      14   2    10   1    13   1                             14      4600    20     169  21   169  22   170  19                            15      1400    61     488  61   450  57   480  55                            16      2100    9      73   9    72   9    87   10                            17      300     1      17   2    25   3    42   5                             18      300     1      11   1    15   2    25   3                             19      200     1      3         5    1    5    1                             20                     5    1    7    1    8    1                             21                     2         4    1    4                                  22                                                                            23                                                                            24                                                                            25                                                                            26                                                                            27                                                                            28                                                                            29                                                                            30                                                                            31                                                                            32                                                                            33                                                                            34                                                                            35                                                                            36                                                                            37                                                                            38                                                                            ______________________________________                                    

                                      TABLE IVC                                   __________________________________________________________________________    Assay of [.sup.57 Co] B.sub.12 and plasma B.sub.12 after elution from         paper chromatography.                                                         All assays were performed at pH 9.0.                                                       B.sub.12 assayed with various binders                                                               Hog IFC +                                                     Hog IFC +                                                                            Hog IFC +                                                                              [3,5,6-Me.sub.3 BZA]                                                                  Hog IFC                            [.sup.57 Co] B.sub.12                                                                      Hog IFC                                                                             Cobinamide                                                                           CN-Cbl[bde-OH]                                                                         (CN, OH)Cba                                                                           Digested                           Fraction #                                                                          (cpm)                                                                             (%)                                                                              (ng)                                                                             (%)                                                                              (ng)                                                                              (%)                                                                              (ng) (%) (ng)                                                                              (%) (ng)                                                                             (%)                             __________________________________________________________________________    1            1                                                                3                                                                             4            1                         1                                      5            2            1    1       1                                      6            2            1        1       1                                  7            3            1        1       2                                  8            3            1        1       2                                  9            5  1  1      1        2       3                                  10    300 1  16 2  4      5    1   7   1   9  1                               11    500 2  26 3  10  1  10   1   13  2   15 2                               12    300 1  19 2  8   1  8    1   9   1   11 1                               13    200 1  22 2  9   1  10   1   12  2   14 2                               14    4600                                                                              20 180                                                                              17 174 21 170  22  148 20  153                                                                              19                              15    14000                                                                             61 474                                                                              46 474 58 435  55  396 53  402                                                                              50                              16    2100                                                                              9  91 9  83  10 81   10  80  11  84 10                              17    300 1  53 5  25  3  27   3   34  4   36 4                               18    300 1  45 4  16  2  19   2   21  3   27 3                               19    200 1  15 1  4      4    1   6   1   9  1                               20           24 2  7   1  7    1   11  1   13 2                               21           21 2  3      4    1   7   1   9  1                               22           5  1                  1       2                                  23           4                     1       1                                  24           3                             1                                  25           4                             1                                  26           5  1                          2                                  27           2                                                                28           1                             1                                  29           2                                                                30           1                                                                31                                                                            32                                                                            33                                                                            34                                                                            35                                                                            36                                                                            37                                                                            38                                                                            __________________________________________________________________________

                  TABLE IVD                                                       ______________________________________                                        Assay of [.sup.57 Co] B.sub.12 and native B.sub.12                            after elution from paper chromatography.                                      All assays were performed at pH 9.0.                                                       B.sub.12 assayed with various binders                            [.sup.57 Co] B.sub.12                                                                        Human R   Human IF  Hog IFC                                    Fraction #                                                                            (cpm)   (%)    (ng) (%)  (ng) (%)  (ng) (%)                           ______________________________________                                        2                                                                             3                                                                             4                                                                             5                                                                             6                                                                             7                                                                             8                                                                             9                                                                             10      200     1      1                   1                                  11      600     2      1         1         2    1                             12      400     1      1         1         2    1                             13      400     1      4    1    3    1    6    2                             14      6800    24     86   25   86   26   90   27                            15      15800   56     210  60   198  60   189  56                            16      3100    11     40   12   38   12   40   12                            17      200     1      2    1    1         3    1                             18      200     1      2    1    1         2    1                             19      100            1                   2    1                             20                                                                            21                                                                            22                                                                            23                                                                            24                                                                            25                                                                            26                                                                            27                                                                            28                                                                            29                                                                            30                                                                            31                                                                            32                                                                            33                                                                            34                                                                            35                                                                            36                                                                            37                                                                            38                                                                            ______________________________________                                    

                                      TABLE IVE                                   __________________________________________________________________________    Summary of the data in Tables IVA- IVD involving assays of [.sup.57 Co]       B.sub.12 and B.sub.12 after elution                                           from paper chromatography. Assays for B.sub.12 were performed at pH 9.0       except where indicated                                                                                    Chromatogram Fractions                                         Item  Assay    1-13  14-16 17-38 1-38                            Sample Chromatographed                                                                     Assayed                                                                             Binder   (ng)                                                                             (%)                                                                              (ng)                                                                             (%)                                                                              (ng)                                                                             (%)                                                                              (ng)                                                                             (%)                          __________________________________________________________________________    [.sup.57 Co] B.sub.12 + B.sub.12                                                           [.sup.57 Co] B.sub.12                                                               --       -- 5  -- 92 -- 2  -- 100                          "            B.sub.12                                                                            Human R  7  2  336                                                                              97 5  1  348                                                                              100                          "            B.sub.12                                                                            Human IF 5  1  322                                                                              98 2  1  329                                                                              100                          "            B.sub.12                                                                            Hog IFC  11 3  319                                                                              95 7  2  337                                                                              100                          [.sup.57 Co] B.sub.12 + plasma B.sub.12                                                    [.sup. 57 Co] B.sub.12                                                              --       -- 6  -- 91 -- 3  -- 100                          "            B.sub.12                                                                            Human R  145                                                                              11 874                                                                              65 324                                                                              24 1343                                                                             100                          "            B.sub.12                                                                            Human R  179                                                                              12 972                                                                              68 283                                                                              20 1434                                                                             100                                             (pH 1.8)                                                   "            B.sub.12                                                                            Hog R    138                                                                              12 721                                                                              64 274                                                                              24 1133                                                                             100                          "            B.sub.12                                                                            Hog R    199                                                                              13 938                                                                              61 402                                                                              26 1539                                                                             100                                             (pH 1.8)                                                   "            B.sub.12                                                                            Hog IFC  100                                                                              10 745                                                                              72 185                                                                              18 1030                                                                             100                          "            B.sub.12                                                                            Human IF 31 4  730                                                                              91 38 5  799                                                                              100                          "            B.sub.12                                                                            Hog IF   37 5  691                                                                              88 56 7  784                                                                              100                          "            B.sub.12                                                                            Rabbit IF                                                                              53 6  737                                                                              84 84 10 874                                                                              100                          "            B.sub.12                                                                            Hog IFC +                                                                              32 4  731                                                                              89 54 7  818                                                                              100                                             Cobinamide                                                 "            B.sub.12                                                                            Hog IFC +                                                                              47 6  624                                                                              83 81 11 752                                                                              100                                             CN-Cbl[bde-OH]                                             "            B.sub.12                                                                            Hog IFC +                                                                              38 5  686                                                                              87 61 8  785                                                                              100                                             [3,5,6-Me.sub.3 BZA]                                                          (CN,OH)Cba                                                 "            B.sub.12                                                                            Hog IFC -                                                                              60 7  639                                                                              80 102                                                                              13 801                                                                              100                                             Digested                                                   __________________________________________________________________________

Referring to the control chromatogram of Table IVD, it is seen that theseveral RID assays of pure vitamin B₁₂ performed variously with human Rprotein, human IF protein and hog IFC gave substantially a singlesymmetrical peak of activity. In each instance greater than 95% of thevitamin B₁₂ was found to be present in fraction 14 through 16. Similarresults, as shown in Table IVB, were obtained from the paperchromatogram of the plasma extract when the binding protein was humanIF, hog IF and rabbit IF. These data are an indication that these threeIF binding proteins are substantially specific in their binding abilityto vitamin B₁₂, and substantially non-reactive with vitamin B₁₂analogues present in plasma.

Efforts were made to modify hog IFC, which is a commonly used binder inRID assays and which has been found to contain as much as 90% hog Rprotein and as little as 10% hog IF, by removing or inactivating the hogR. In several instances the hog IFC was incubated with an excess amountof three chemically synthesized vitamin B₁₂ analogues before it wasutilized in the RID assay. Referring to Table IVC, it is seen that afterthis modification the chromatogram results obtained utilizing themodified hog IFC closely resemble the results obtained withsubstantially pure hog IF. It is therefore seen, that in the practice ofthe present invention, mixtures of protein including both vitamin B₁₂specific binding protein and binding protein which is not specific tovitamin B₁₂ can be modified by the addition of an excess amount ofvitamin B₁₂ analogue, by which process the analogue binds with thenon-specific protein to render it substantially bound or inactive sothat it is not available to react with vitamin B₁₂ or (⁵⁷ Co) B₁₂present in samples undergoing RID tests. The amount of vitamin B₁₂analogue to be added to a mixture of specific and non-specific proteinsin order to bind or inactivate the non-specific proteins may vary over awide range, depending on both the proteins which are present and thevitamin B₁₂ analogues which are utilized as the binding or inactivatingmaterial. Generally speaking, for the examples shown in Table IVC,cobinamide may be added in an amount equal to that required for completebinding of the non-specific protein, up to an amount as much as tenmillion times greater than the amount needed to bind the protein, withthe preferred range being about ten to about ten thousand times inexcess of that required for complete binding. CN-Cbl (bde-OH), known asCoB-cyano-cobamic a,c,g-triamide may be utilized in an amount at leastabout ten times to about ten million times in excess of the amountrequired to bind with the non-specific protein, with an amount of aboutone hundred to about one hundred thousand times excess being preferred.The (3,5,6-Me₃ BZA) (CN,OH)-cba, known asCoα(3,5,6-trimethylbenzimidazole) cobamide should also be utilized inamounts from about one to ten million times in excess of the amount ofnon-specific protein, with an amount in the range of about ten to aboutten thousand times excess being preferred. Suitable amounts of othervitamin B₁₂ analogues may be utilized in a similar manner to bind orinactivate non-specific proteins present in mixtures with specificproteins in order to obtain a preparation of binding protein which willsubstantially bind only ⁵⁷ Co-B₁₂, or the vitamin B₁₂ naturally presentin the samples being tested, and thus give a more accurate quantitativeRID assay of vitamin B₁₂ in samples undergoing tests.

Again, referring to Table IVC, data on samples of hog IFC digested withtrypsin and chymotrypsin are shown. These and other proteolytic enzymesare specific in their ability to substantially digest R proteins whileleaving intrinsic factor proteins unaffected and available assubstantially the only protein in for binding ⁵⁷ CO-B₁₂ and vitamin B₁₂in RID assays. Other enzymes, including, for example, elastase may beutilized for the same purpose. The amount of the enzymes utilized is inthe range of about 0.01 to about 100 milligrams per mililiter of proteintreated, with a preferred amount being about 0.05 to about 40 milligramsper mililiter of protein. Utilizing this proteolytic enzyme digestionprocess a protein binder is provided which substantially binds only ⁵⁷Co-B₁₂ and vitamin B₁₂ and is not affected by vitamin B₁₂ analogues inthe samples being tested and which therefore gives a more accurate RIDassay than is obtained when utilizing the original mixture of hog IFCproteins which included non-specific proteins which would have beencapable of reacting with the newly discovered vitamin B₁₂ analogues insamples to give inaccurate assays as to the amount of vitamin B₁₂ intest samples.

Now referring to Table IVA, when samples from the plasma extractthromatogram were assayed for vitamin B₁₂ with human R, hog R and hogIFC, different results were obtained than when those samples wereassayed with human IF, hog IF, rabbit IF or hog IFC treated with vitaminB₁₂ analogues or hog IFC digested with proteolytic enzymes. In each casewhere human R, hog R or untreated hog IFC were utilized as the bindingprotein the tests gave the appearance that more vitamin B₁₂ was presentin the chromatogram samples, especially in fractions 1 through 13 and 17through 38. This observation, when taken with the above data, providesstrong evidence that normal human plasma contains a number of vitaminB₁₂ analogues that compete with ⁵⁷ Co-B₁₂, in significant amounts, forbinding to R protein. It also indicates that such activity on the partof the B₁₂ analogues is substantially absent when the binding proteinutilized in the RID assay is substantially specific to vitamin B₁₂.

It should also be noted, see Table IVA, that the chromatogram datasuggests that the lack of specificity of human R and hog R is unchangedwhen RID assays are performed at acid pH. This indicates that erroneousresults will be obtained for the true vitamin B₁₂ content of sampleswhich contain vitamin B₁₂ analogues when RID assays are performed atacid pH.

Using the same techniques and criteria described above it has beendiscovered that vitamin B₁₂ analogues are not only present in serumobtained from human blood, but are also present in mammalian tissues ineven higher concentrations than they are in blood. Vitamin B₁₂ analoguesextracted from mammalian tissues have been purified using the sameschemes as described above. When analyzed utilizing paperchromatography, they exhibited similar mobilities to those of thevitamin B₁₂ analogues observed in the samples extracted from bloodserum. Since larger amounts of the vitamin B₁₂ analogues are present intissue, they can be observed visually as red or orange spots duringpaper chromatography. The absorption spectra of vitamin B₁₂ analoguespurified from tissue extracts have been determined and demonstrate thatthey are similar to, but distinct from, the absorption spectrum of truevitamin B₁₂. These observations provide additional evidence that thematerials in blood serum which preferentially react with R proteins andnot intrinsic factor proteins are in fact varieties of vitamin B₁₂analogues.

The newly discovered vitamin B₁₂ analogues also differ from vitamin B₁₂in terms of their biological activity. Thus, as shown in Table V, theserum vitamin B₁₂ values obtained with Euglena gracilis for elevenpatients diagnosed to have vitamin B₁₂ deficiency were substantiallysimilar to the results obtained by RID assay using human IF or hog IF asthe binding protein.

                  TABLE V                                                         ______________________________________                                        Vitamin B.sub.12 levels in 21 patients with clinical evidence                 of vitamin B.sub.12 deficiency                                                                            Vitamin                                                                       B.sub.12                                                                      assayed                                           Vitamin B.sub.12 assayed with various binders                                                             Euglena                                                  Human R  Hog R    Human IF                                                                              Hog IF Gracilis                              Patient                                                                              (pg/ml)  (pg/ml)  (pg/ml) (pg/ml)                                                                              (pg/ml)                               ______________________________________                                        1      155      138      16       0     43                                    2      310      295      51      86     52                                    3      310      255      24      35     46                                    4      135      132       0      22     40                                    5      215      250      52      60     25                                    6      347      342       0       0     56                                    7      102      120       0       0     35                                    8      240      242      58      65     79                                    9      160      125      38      23      0                                    10      85       85       0      12     23                                    11     235      255       5      11      0                                    12     188      190      41      38                                           13     178      192      48      38                                           14     298      305      53      57                                           15     355      310      78      82                                           16     178      210      48      22                                           17     128      140      31       8                                           18     106      190      50      40                                           19     163      155       0       0                                           20     178      132      25       0                                           21     230      215      75      41                                           Mean                                                                          (1- 11)                                                                              209      203      22      29     36                                    Mean                                                                          (1-21) 205      204      33      30                                           Normal                                                                        Range                                                                         (mean ±                                                                    2 std.                                                                        dev.)  203-1482 197-1493 136-656 157-717                                                                              (>130)                                Number                                                                        within                                                                        normal                                                                        range  9 (43%)  10 (48%)  0       0     0                                     ______________________________________                                    

It is to be further noted, that all of the values obtained by eithermicrobiologic assay or by assay using human IF or hog IF weresubstantially lower than the values obtained when the RID assay wascarried out utilizing human R or hog R as the binding protein. Thisindicates that the vitamin B₁₂ analogues which have now been identifiedin mammalian blood and tissue do not possess vitamin B₁₂ activity of thetype which is required to promote the growth of Euglena gracilis.

Data was obtained on ten additional patients diagnosed to be vitamin B₁₂deficient and the total of 21 patients with vitamin B₁₂ deficiency areshown in Table V. In each of the 21 patients the vitamin B₁₂ valuesfound when the RID assay was carried out utilizing human IF or hog IFwere below the range of vitamin B₁₂ values found in a control group of74 normal subjects. However, when the RID assay was carried oututilizing human R or hog R only about half of the 21 vitamin B₁₂deficient patients were found to assay below the range of normalsubjects for vitamin B₁₂. This indicates that where the newly foundvitamin B₁₂ analogues are present in the samples being tested, and thebinding protein is not specific to vitamin B₁₂, the resulting assays maysuggest that a truly vitamin B₁₂ deficient patient is not within thedeficient range. This may lead to delay of treatment of that patient forvitamin B₁₂ deficiency. It also indicates that the vitamin B₁₂ analoguesthat have now been discovered lack the therapeutic or beneficialactivity of vitamin B₁₂ in the sense of being unable to prevent thehematologic and/or neurologic abnormalities associated with vitamin B₁₂deficiency.

                  TABLE VI                                                        ______________________________________                                        Analysis of vitamin B.sub.12 binders and assay pH used in commercial          kits sold for the assay of vitamin B.sub.12 in clinical laboratories                          Vitamin B.sub.12                                                              Binding Protein                                                                 intrinsic                                                                              R                                                                    factor.sup.(a)                                                                         protein.sup.(b)                                                                        Assay pH                                  Source of Binder  (%)      (%)      of Kit.sup.(d)                            ______________________________________                                        Hog IF            97        0       --                                        Hog R              0       98       --                                        Hog IFC           25       75       --                                        Diagnostic Products Corp. Kit                                                                   35       60       1.7                                       New England Nuclear Kit                                                                         20       82       4.1                                       Bio-Rad Laboratories Kit                                                                        30       71       1.9                                       Medvak Diagnostic Products Kit                                                                  13       85       1.8                                       Schwarz/Mann Kit  34       67       9.1                                       Pharmacia Diagnostics Kit                                                                       1 (33).sup.(c)                                                                         67       4.1                                       ______________________________________                                         .sup.(a) Inhibition of [.sup.57 Co] B.sub.12 binding observed with antiIF     antibodies at pH 7.5.                                                         .sup.(b) Inhibition of [.sup.57 Co] B.sub.12 binding observed with a 100      fold molar excess of cobinamide at pH 7.5.                                    .sup.(c) In this kit the binder is covalently attached to an insoluble        matrix and because of steric factors it may not be accesible to               antiintrinsic factor antibodies. Thus the value for % intrinsic factor ma     be as high as 33%.                                                            .sup.(d) Refers to the pH measured after the addition of all of the           components of each individual kit. R protein retains its full B.sub.12        binding ability over the range of pH 1.7-9.1 but intrinsic factor loses       10% and 99% of its B.sub.12 binding ability at pH 4.1 and 1.9,                respectively. Thus kits that use pH 4.1 have slightly less % intrinsic        factor than is indicated in the table while those that use pH 1.7-1.9 are     employing essentially no intrinsic factor.                               

There are many commercial RID assay type kits available for the assay ofvitamin B₁₂ in clinical laboratories. Table VI sets forth an analysis ofseveral such kits, and a comparison of the types of protein found inthose kits with hog IF, hog R and hog IFC. By reference to Table VI, itis seen that the commercial kits appear to have only about 13% to about35% intrinsic factor and from about 60% to about 85% R protein. It istherefore suspected, that the use of these kits will give substantiallyerroneous assays of the amount of vitamin B₁₂ present in a sample whenthe sample also includes vitamin B₁₂ analogues, such as those newlydiscovered to exist in mammalian blood and tissues. It has also beendetermined that the effectiveness of intrinsic factor to bind vitaminB₁₂ is somewhat pH dependent, with intrinsic factor losing about 10% ofits binding ability at a pH of about 4.1 and losing about 99% of itsbinding ability at a pH of about 1.9. Thus, to the extent that thecommercial kits use a pH of about 4.1 during binding they would haveabout 10% less intrinsic factor than shown in Table VI. Those kitshaving a pH during binding of about 1.7 to about 1.9 would obtainsubstantially no binding from intrinsic factor.

While the invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that the foregoing and other modifications orchanges in form and details may be made therein without departing fromthe spirit and scope of the invention.

What is claimed is:
 1. A radioisotope dilution assay method of measuringthe vitamin B₁₂ (cobalamin) level in a sample which may also containvitamin B₁₂ analogue comprising: contacting said sample with a knownamount of radioisotope of vitamin B₁₂ and with an assay composition,said assay composition including an active component of binding proteinwhich is substantially specific to vitamin B₁₂ and substantiallynon-reactive with vitamin B₁₂ analogue, wherein one or more bindingprotein which is non-specific to vitamin B₁₂ may be present in theprecursor of said assay composition and wherein any said non-specificbinding protein has been rendered substantially inactive or inoperativeas to its ability to bind with vitamin B₁₂, said assay composition beingsubstantially free of any other substance which is present in a form inwhich it can react with vitamin B₁₂ and any vitamin B₁₂ analogue whichmay be present in said to-be-assayed sample.
 2. The process of claim 1wherein the non-specific binding protein has been treated to render itsubstantially inactive or inoperative as to its ability to bind withvitamin B₁₂ by the process of treating said non-specific binding proteinwith one or more vitamin B₁₂ analogue in an amount sufficient to rendersaid non-specific binding protein inactive or inoperative as to itsability to bind with vitamin B₁₂.
 3. The process of claim 2 wherein atleast a portion of said vitamin B₁₂ analogue used to treat saidnon-specific binding protein is selected from the group of vitamin B₁₂analogues consisting of cobinamide, CN-Cbl(bde-OH) and (3,5,6-Me₃BZA)(CN,OH) Cba.
 4. The process of claim 2 wherein said binding proteinis treated with one or more proteolytic enzyme in an amount sufficientto render it substantially inactive or inoperative as to its ability tobind with vitamin B₁₂ and wherein said enzyme is substantiallynon-reactive with said binding protein material which is a substantiallyspecific binder to vitamin B₁₂.
 5. The process of claim 4 wherein thespecific active binder component includes intrinsic factor protein andthe non-specific protein binder includes R protein.
 6. The process ofclaim 4 wherein at least a portion of the proteolytic enzyme is selectedfrom the group consisting of trypsin, chymotrypsin and elastase.
 7. Theprocess of claim 4 wherein the proteolytic enzyme is present in theamount of about 0.01 mg/ml to about 100 mg/ml, based on the mililitervolume of the assay composition precursor.
 8. The process of claim 6wherein the proteolytic enzyme is present in the amount of about 0.05mg/ml to about 40 mg/ml, based on the mililiter volume of the assaycomposition precursor.
 9. A Composition for use in the assay of vitaminB₁₂ (cobalamin) in a sample which may also contain vitamin B₁₂ analogue,said assay composition including: an active component of binding proteinwhich is substantially specific to vitamin B₁₂ and substantiallynon-reactive with vitamin B₁₂ analogue, said assay composition alsoincluding vitamin B₁₂ analogue and non-specific binding protein bound tosaid vitamin B₁₂ analogue, said vitamin B₁₂ analogue being present insaid assay composition in an amount sufficient to render saidnon-specific binding protein substantially inactive or inoperative as toits ability to bind with vitamin B₁₂, said assay composition beingsubstantially free of other substances which are present in a form inwhich they can react with vitamin B₁₂ and any vitamin B₁₂ analogue whichmay be present in said to-be-assayed sample.
 10. The composition ofclaim 9 wherein said specific active binding protein includes intrinsicfactor protein and the said non-specific binding protein, which has beentreated with vitamin B₁₂ analogues to render it substantially inactiveor inoperative includes R protein.
 11. The composition of claim 10wherein at least a portion of said vitamin B₁₂ analogue used to treatsaid non-specific binding protein is selected from the group consistingof cobinamide, CN-Cbl(bde-OH) and (3,5,6-Me₃ BZA)(CN,OH)Cba.
 12. Theassay composition of claim 9 wherein vitamin B₁₂ analogue used to treatsaid non-specific binding protein is present in the amount of about1-fold to about a 100,000-fold molar excess, based on the molar amountof said non-specific protein in said assay composition.
 13. Thecomposition of claim 8 wherein said vitamin B₁₂ analogue used to treatsaid non-specific binding protein is present in the amount of about a10-fold to about 10,000-fold molar excess based on the molar of saidnon-specific protein in said assay composition.
 14. The method ofpreparing the assay composition of claim 9 wherein a precursor mixtureincluding binding material substantially specific to vitamin B₁₂ andbinding material which is non-specific to vitamin B₁₂ is treated withsufficient vitamin B₁₂ analogue to inactivate or render inoperativesubstantially all of said binding material which is non-specific tovitamin B₁₂, said vitamin B₁₂ analogue being substantially reactive withsaid non-specific binder and substantially non-reactive with thematerial which is a specific binder for vitamin B₁₂.
 15. The method ofclaim 14 wherein in said precussor mixture said specific active binderfor vitamin B₁₂ includes intrinsic factor binding protein and saidbinding material which is non-specific to vitamin B₁₂ includes R bindingprotein.
 16. The method of claim 15 wherein at least a portion of saidvitamin B₁₂ analogue used to treat said non-specific binding protein insaid precursor mixture is selected from the group consisting ofcobinamide, CN-Cbl(bde-OH) and (3,5,6-Me₃ BZA)(CN,OH) Cba.
 17. Themethod of claim 15 wherein said vitamin B₁₂ analogue used to treat saidnon-specific binding protein in said precursor mixture is present in theamount of about 1-fold to about a 100,000-fold molar excess, based onthe molar amount of the said non-specific protein in said assaycomposition.
 18. The method of claim 15 wherein said vitamin B₁₂analogue used to treat said non-specific binding protein in saidprecursor mixture is present in the amount of about a 10-fold to about10,000 fold molar excess based on the molar amount of said non-specificprotein in said assay composition.
 19. An assay method for measuring thevitamin B₁₂ (cobalamin) level in a sample which may also contain vitaminB₁₂ analogue, including the step of: contacting said sample with anassay composition, said assay composition including an active componentwhich is substantially specific to vitamin B₁₂ in said assay andsubstantially non-reactive in said assay with vitamin B₁₂ analogue whichmay be present in said sample, said assay composition also beingsubstantially free of any other composition which is present in a formin which it can react in said assay with material selected from thegroup consisting of vitamin B₁₂ analogue and both vitamin B₁₂ andvitamin B₁₂ analogue.
 20. The method of claim 19 wherein said activecomponent includes binding protein.
 21. The method of claim 20 whereinsaid binding protein is intrinsic factor.
 22. The method of claim 19wherein one or more non-specific composition may be present in theprecursor of said assay composition, said non-specific composition beingcapable of reacting with material selected from the group consisting ofvitamin B₁₂ analogue and both vitamin B₁₂ and vitamin B₁₂ analogue, andwherein any said non-specific composition present in said precursor hasbeen rendered substantially inactive or inoperative as to its ability,in said assay, to react with material selected from the group consistingof vitamin B₁₂ analogue and both vitamin B₁₂ and vitamin B₁₂ analogue.23. The method of claim 22 wherein said specific active binder forvitamin B₁₂ includes intrinsic factor binding protein and saidnon-specific composition includes R binding protein.
 24. The compositionof claim 10 wherein at least a portion of said Vitamin B₁₂ analogue usedto treat said non-specific binding protein is cobinamide.
 25. Acomposition for use in the assay of vitamin B₁₂ (cobalamin) wherein theactive component is substantially free of substances which react withvitamin B₁₂ analogues.